Discovery of novel 1H-benzo[d]imidazole-4,7-dione based transglutaminase 2 inhibitors as p53 stabilizing anticancer agents in renal cell carcinoma.

Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure-activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the ß-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the ß-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.

Integration of Hybridization Strategies in Pyridine-Urea Scaffolds for Novel Anticancer Agents: Design, Synthesis, and Mechanistic Insights.

Annually, millions of new cancer cases are reported, leading to millions of deaths worldwide. Among the newly reported cases, breast and colon cancers prevail as the most frequently detected variations. To effectively counteract this rapid increase, the development of innovative therapies is crucial. Small molecules possessing pyridine and urea moieties have been reported in many of the currently available anticancer agents, especially VEGFR2 inhibitors. With this in mind, a rational design approach was employed to create hybrid small molecules combining urea and pyridine. These synthesized compounds underwent in vitro testing against breast and colon cancer cell lines, revealing potent submicromolar anticancer activity. Compound 8a, specifically, exhibited an impressive GI50 value of 0.06 µM against the MCF7 cancer cell line, while compound 8h displayed the highest cytotoxic activity against the HCT116 cell line, with a GI50 of 0.33 ± 0.042 µM. Notably, compounds 8a, 8h, and 8i demonstrated excellent safety profiles when tested on normal cells. Molecular docking, dynamic studies, and free energy calculations were employed to validate the affinity of these compounds as VEGFR2 inhibitors.

Design and Synthesis of a Novel 4-aryl-N-(2-alkoxythieno [2,3-b]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide DGG200064 Showed Therapeutic Effect on Colon Cancer through G2/M Arrest.

Cancer cells are characterized by an abnormal cell cycle. Therefore, the cell cycle has been a potential target for cancer therapeutic agents. We developed a new lead compound, DGG200064 (7c) with a 2-alkoxythieno [2,3-b]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide core skeleton. To evaluate its properties, compound DGG200064 was tested in vivo through a xenograft mouse model of colorectal cancer using HCT116 cells. The in vivo results showed high cell growth inhibition efficacy. Our results confirmed that the newly synthesized DGG200064 inhibits the growth of colorectal cancer cells by inducing G2/M arrest. Unlike the known cell cycle inhibitors, DGG200064 (GI50 = 12 nM in an HCT116 cell-based assay) induced G2/M arrest by selectively inhibiting the interaction of FBXW7 and c-Jun proteins. Additionally, the physicochemical properties of the lead compounds were analyzed. Based on the results of the study, we suggested further development of DGG200064 as a novel oral anti-colorectal cancer drug.

Reply to Krupenko et al. Comment on "Lee et al. The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer Cancers 2020, 12, 1382".

In the Cancers paper, we observed the increase ALDH1L1 protein expression following oncogenesis, as well as a therapeutic effect, by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC) [...].

Oxoglutarate Carrier Inhibition Reduced Melanoma Growth and Invasion by Reducing ATP Production.

Recent findings indicate that (a) mitochondria in proliferating cancer cells are functional, (b) cancer cells use more oxygen than normal cells for oxidative phosphorylation, and (c) cancer cells critically rely on cytosolic NADH transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. In a spontaneous lung cancer model, tumor growth was reduced by 50% in heterozygous oxoglutarate carrier (OGC) knock-out mice compared with wild-type counterparts. To determine the mechanism through which OGC promotes tumor growth, the effects of the OGC inhibitor N-phenylmaleimide (NPM) on mitochondrial activity, oxygen consumption, and ATP production were evaluated in melanoma cell lines. NPM suppressed oxygen consumption and decreased ATP production in melanoma cells in a dose-dependent manner. NPM also reduced the proliferation of melanoma cells. To test the effects of NPM on tumor growth and metastasis in vivo, NPM was administered in a human melanoma xenograft model. NPM reduced tumor growth by approximately 50% and reduced melanoma invasion by 70% at a dose of 20 mg/kg. Therefore, blocking OGC activity may be a useful approach for cancer therapy.

ATP Production Relies on Fatty Acid Oxidation Rather than Glycolysis in Pancreatic Ductal Adenocarcinoma.

Glycolysis is known as the main pathway for ATP production in cancer cells. However, in cancer cells, glucose deprivation for 24 h does not reduce ATP levels, whereas it does suppress lactate production. In this study, metabolic pathways were blocked to identify the main pathway of ATP production in pancreatic ductal adenocarcinoma (PDAC). Blocking fatty acid oxidation (FAO) decreased ATP production by 40% in cancer cells with no effect on normal cells. The effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acids instead of calories. A low-fat diet caused a 70% decrease in pancreatic preneoplastic lesions compared with the control, whereas a high-fat diet caused a two-fold increase in preneoplastic lesions accompanied with increase of ATP production in the Kras (G12D)/Pdx1-cre PDAC model. The present results suggest that ATP production in cancer cells is dependent on FAO rather than on glycolysis, which can be a therapeutic approach by targeting cancer energy metabolism.

Targeting Oxidative Phosphorylation Reverses Drug Resistance in Cancer Cells by Blocking Autophagy Recycling.

The greatest challenge in cancer therapy is posed by drug-resistant recurrence following treatment. Anticancer chemotherapy is largely focused on targeting the rapid proliferation and biosynthesis of cancer cells. This strategy has the potential to trigger autophagy, enabling cancer cell survival through the recycling of molecules and energy essential for biosynthesis, leading to drug resistance. Autophagy recycling contributes amino acids and ATP to restore mTOR complex 1 (mTORC1) activity, which leads to cell survival. However, autophagy with mTORC1 activation can be stalled by reducing the ATP level. We have previously shown that cytosolic NADH production supported by aldehyde dehydrogenase (ALDH) is critical for supplying ATP through oxidative phosphorylation (OxPhos) in cancer cell mitochondria. Inhibitors of the mitochondrial complex I of the OxPhos electron transfer chain and ALDH significantly reduce the ATP level selectively in cancer cells, terminating autophagy triggered by anticancer drug treatment. With the aim of overcoming drug resistance, we investigated combining the inhibition of mitochondrial complex I, using phenformin, and ALDH, using gossypol, with anticancer drug treatment. Here, we show that OxPhos targeting combined with anticancer drugs acts synergistically to enhance the anticancer effect in mouse xenograft models of various cancers, which suggests a potential therapeutic approach for drug-resistant cancer.

Transglutaminase 2-Mediated p53 Depletion Promotes Angiogenesis by Increasing HIF-1α-p300 Binding in Renal Cell Carcinoma.

Angiogenesis and the expression of vascular endothelial growth factor (VEGF) are increased in renal cell carcinoma (RCC). Transglutaminase 2 (TGase 2), which promotes angiogenesis in endothelial cells during wound healing, is upregulated in RCC. Tumor angiogenesis involves three domains: cancer cells, the extracellular matrix, and endothelial cells. TGase 2 stabilizes VEGF in the extracellular matrix and promotes VEGFR-2 nuclear translocation in endothelial cells. However, the role of TGase 2 in angiogenesis in the cancer cell domain remains unclear. Hypoxia-inducible factor (HIF)-1α-mediated VEGF production underlies the induction of angiogenesis in cancer cells. In this study, we show that p53 downregulated HIF-1α in RCC, and p53 overexpression decreased VEGF production. Increased TGase 2 promoted angiogenesis by inducing p53 degradation, leading to the activation of HIF-1α. The interaction of HIF-1α and p53 with the cofactor p300 is required for stable transcriptional activation. We found that TGase 2-mediated p53 depletion increased the availability of p300 for HIF-1α-p300 binding. A preclinical xenograft model suggested that TGase 2 inhibition can reverse angiogenesis in RCC.

Inhibition of Transglutaminase 2 but Not of MDM2 Has a Significant Therapeutic Effect on Renal Cell Carcinoma.

More than 50% of human cancers harbor TP53 mutations and increased expression of Mouse double minute 2 homolog(MDM2), which contribute to cancer progression and drug resistance. Renal cell carcinoma (RCC) has an unusually high incidence of wild-type p53, with a mutation rate of less than 4%. MDM2 is master regulator of apoptosis in cancer cells, which is triggered through proteasomal degradation of wild-type p53. Recently, we found that p53 protein levels in RCC are regulated by autophagic degradation. Transglutaminase 2 (TGase 2) was responsible for p53 degradation through this pathway. Knocking down TGase 2 increased p53-mediated apoptosis in RCC. Therefore, we asked whether depleting p53 from RCC cells occurs via MDM2-mediated proteasomal degradation or via TGase 2-mediated autophagic degradation. In vitro gene knockdown experiments revealed that stability of p53 in RCC was inversely related to levels of both MDM2 and TGase 2 protein. Therefore, we examined the therapeutic efficacy of inhibitors of TGase 2 and MDM2 in an in vivo model of RCC. The results showed that inhibiting TGase 2 but not MDM2 had efficient anticancer effects.

The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer.

Lung adenocarcinoma cells express high levels of ALDH1L1, an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and NAD(P)H. In this study, we evaluated the potential of ALDH1L1 as a therapeutic target by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC). Reporter assays revealed KRAS-mediated upregulation of the ALDH1L1 promoter in human NSCLC cells. Aldh1l1-/- mice exhibited a normal phenotype, with a 10% decrease in Kras-driven lung tumorigenesis. By contrast, the inhibition of oxidative phosphorylation inhibition using phenformin in Aldh1l1-/-; KrasLA2 mice dramatically decreased the number of tumor nodules and tumor area by up to 50%. Furthermore, combined treatment with pan-ALDH inhibitor and phenformin showed a decreased number and area of lung tumors by 70% in the KrasLA2 lung cancer model. Consistent with this, previous work showed that the combination of ALDH1L1 knockdown and phenformin treatment decreased ATP production by as much as 70% in NSCLS cell lines. Taken together, these results suggest that the combined inhibition of ALDH activity and oxidative phosphorylation represents a promising therapeutic strategy for NSCLC.

Gastric cancer depends on aldehyde dehydrogenase 3A1 for fatty acid oxidation.

The major source of ATP in cancer cells remains unclear. Here, we examined energy metabolism in gastric cancer cells and found increased fatty acid oxidation and increased expression of ALDH3A1. Metabolic analysis showed that lipid peroxidation by reactive oxygen species led to spontaneous production of 4-hydroxynonenal, which was converted to fatty acids with NADH production by ALDH3A1, resulting in further fatty acid oxidation. Inhibition of ALDH3A1 by knock down using siRNA of ALDH3A1 resulted in significantly reduced ATP production by cancer cells, leading to apoptosis. Oxidative phosphorylation by mitochondria in gastric cancer cells was driven by NADH supplied via fatty acid oxidation. Therefore, blockade of ALDH3A1 together with mitochondrial complex I using gossypol and phenformin led to significant therapeutic effects in a preclinical gastric cancer model.

Loss of SLC25A11 causes suppression of NSCLC and melanoma tumor formation.

BACKGROUND: Fast growing cancer cells require greater amounts of ATP than normal cells. Although glycolysis was suggested as a source of anabolic metabolism based on lactate production, the main source of ATP to support cancer cell metabolism remains unidentified. METHODS: We have proposed that the oxoglutarate carrier SLC25A11 is important for ATP production in cancer by NADH transportation from the cytosol to mitochondria as a malate. We have examined not only changes of ATP and NADH but also changes of metabolites after SLC25A11 knock down in cancer cells. FINDINGS: The mitochondrial electron transport chain was functionally active in cancer cells. The cytosolic to mitochondrial NADH ratio was higher in non-small cell lung cancer (NSCLC) and melanoma cells than in normal cells. This was consistent with higher levels of the oxoglutarate carrier SLC25A11. Blocking malate transport by knockdown of SLC25A11 significantly impaired ATP production and inhibited the growth of cancer cells, which was not observed in normal cells. In in vivo experiments, heterozygote of SLC25A11 knock out mice suppressed KRASLA2 lung tumor formation by cross breeding. INTERPRETATION: Cancer cells critically depended on the oxoglutarate carrier SLC25A11 for transporting NADH from cytosol to mitochondria as a malate form for the purpose of ATP production. Therefore blocking SLC25A11 may have an advantage in stopping cancer growth by reducing ATP production. FUND: The Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT to SYK (NRF-2017R1A2B2003428).

Transglutaminase 2 Promotes Autophagy by LC3 Induction through p53 Depletion in Cancer Cell.

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.

Renal Cell Carcinoma Is Abrogated by p53 Stabilization through Transglutaminase 2 Inhibition.

In general, expression of transglutaminase 2 (TGase 2) is upregulated in renal cell carcinoma (RCC), resulting in p53 instability. Previous studies show that TGase 2 binds to p53 and transports it to the autophagosome. Knockdown or inhibition of TGase 2 in RCC induces p53-mediated apoptosis. Here, we screened a chemical library for TGase 2 inhibitors and identified streptonigrin as a potential therapeutic compound for RCC. Surface plasmon resonance and mass spectroscopy were used to measure streptonigrin binding to TGase 2. Mass spectrometry analysis revealed that streptonigrin binds to the N-terminus of TGase 2 (amino acids 95⁻116), which is associated with inhibition of TGase 2 activity in vitro and with p53 stabilization in RCC. The anti-cancer effects of streptonigrin on RCC cell lines were demonstrated in cell proliferation and cell death assays. In addition, a single dose of streptonigrin (0.2 mg/kg) showed marked anti-tumor effects in a preclinical RCC model by stabilizing p53. Inhibition of TGase 2 using streptonigrin increased p53 stability, which resulted in p53-mediated apoptosis of RCC. Thus, targeting TGase 2 may be a new therapeutic approach to RCC.

Targeting Mitochondrial Oxidative Phosphorylation Abrogated Irinotecan Resistance in NSCLC.

Anticancer drug resistance is a major challenge of cancer therapy. We found that irinotecan-resistant NSCLC cells showed increased mitochondrial oxidative phosphorylation compared to the drug sensitive NSCLC cells. Previously, we found that combined inhibition of aldehyde dehydrogenase using gossypol, and mitochondrial complex I using phenformin, effectively reduced oxidative phosphorylation in NSCLC. Here, we showed that targeting oxidative phosphorylation with gossypol and phenformin abrogated irinotecan resistance in NSCLC. Furthermore, irinotecan treatment by blocking oxidative phosphorylation induced synergistic anti-cancer effect in NSCLC. The pre-clinical xenograft model of human NSCLC also demonstrated a therapeutic response to the dual targeting treatment. Therefore, this combination of gossypol and phenformin increases irinotecan sensitivity as well as preventing irinotecan resistance.

Allosteric inhibition site of transglutaminase 2 is unveiled in the N terminus.

Previously we have demonstrated transglutaminase 2 (TGase 2) inhibition abrogated renal cell carcinoma (RCC) using GK921 (3-(phenylethynyl)-2-(2-(pyridin-2-yl)ethoxy)pyrido[3,2-b]pyrazine), although the mechanism of TGase 2 inhibition remains unsolved. Recently, we found that the increase of TGase 2 expression is required for p53 depletion in RCC by transporting the TGase 2 (1-139 a.a)-p53 complex to the autophagosome, through TGase 2 (472-687 a.a) binding p62. In this study, mass analysis revealed that GK921 bound to the N terminus of TGase 2 (81-116 a.a), which stabilized p53 by blocking TGase 2 binding. This suggests that RCC survival can be stopped by p53-induced cell death through blocking the p53-TGase 2 complex formation using GK921. Although GK921 does not bind to the active site of TGase 2, GK921 binding to the N terminus of TGase 2 also inactivated TGase 2 activity through acceleration of non-covalent self-polymerization of TGase 2 via conformational change. This suggests that TGase 2 has an allosteric binding site (81-116 a.a) which changes the conformation of TGase 2 enough to accelerate inactivation through self-polymer formation.

Regulation of bioenergetics through dual inhibition of aldehyde dehydrogenase and mitochondrial complex I suppresses glioblastoma tumorspheres.

Background: Targeted approaches for treating glioblastoma (GBM) attempted to date have consistently failed, highlighting the imperative for treatment strategies that operate on different mechanistic principles. Bioenergetics deprivation has emerged as an effective therapeutic approach for various tumors. We have previously found that cancer cells preferentially utilize cytosolic NADH supplied by aldehyde dehydrogenase (ALDH) for ATP production through oxidative phosphorylation (OxPhos). This study is aimed at examining therapeutic responses and underlying mechanisms of dual inhibition of ALDH and OxPhos against GBM. Methods: For inhibition of ALDH and OxPhos, the corresponding inhibitors, gossypol and phenformin were used. Biological functions, including ATP levels, stemness, invasiveness, and viability, were evaluated in GBM tumorspheres (TSs). Gene expression profiles were analyzed using microarray data. In vivo anticancer efficacy was examined in a mouse orthotopic xenograft model. Results: Combined treatment of GBM TSs with gossypol and phenformin significantly reduced ATP levels, stemness, invasiveness, and cell viability. Consistently, this therapy substantially decreased expression of genes associated with stemness, mesenchymal transition, and invasion in GBM TSs. Supplementation of ATP using malate abrogated these effects, whereas knockdown of ALDH1L1 mimicked them, suggesting that disruption of ALDH-mediated ATP production is a key mechanism of this therapeutic combination. In vivo efficacy confirmed remarkable therapeutic responses to combined treatment with gossypol and phenformin. Conclusion: Our findings suggest that dual inhibition of tumor bioenergetics is a novel and effective strategy for the treatment of GBM.

Aldehyde dehydrogenase is used by cancer cells for energy metabolism.

We found that non-small-cell lung cancer (NSCLC) cells express high levels of multiple aldehyde dehydrogenase (ALDH) isoforms via an informatics analysis of metabolic enzymes in NSCLC and immunohistochemical staining of NSCLC clinical tumor samples. Using a multiple reaction-monitoring mass spectrometry analysis, we found that multiple ALDH isozymes were generally abundant in NSCLC cells compared with their levels in normal IMR-90 human lung cells. As a result of the catalytic reaction mediated by ALDH, NADH is produced as a by-product from the conversion of aldehyde to carboxylic acid. We hypothesized that the NADH produced by ALDH may be a reliable energy source for ATP production in NSCLC. This study revealed that NADH production by ALDH contributes significantly to ATP production in NSCLC. Furthermore, gossypol, a pan-ALDH inhibitor, markedly reduced the level of ATP. Gossypol combined with phenformin synergistically reduced the ATP levels, which efficiently induced cell death following cell cycle arrest.

Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion.

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

Histamine receptor 2-mediated growth-differentiation factor-15 expression is involved in histamine-induced melanogenesis.

Vitiligo is a progressive depigmenting disorder. Histamine has been shown to induce melanogenesis via histamine receptor 2, suggesting the possibility of histamine as a repigmenting agent for the treatment of vitiligo. However, the role and signaling mechanism of histamine are still unclear in melanogenesis, especially in relation to growth-differentiation factor-15, which is a protein belonging to transforming growth factor beta and found to be overexpressed in metastatic or malignant melanoma. We found that histamine induces growth-differentiation factor-15 in melanoma cell lines such as SK-MEL-2, B16F10, and melan-a cells. Therefore, in the present study, the role of growth-differentiation factor-15 in histamine-induced melanogenesis was investigated using gene silencing or overexpression of growth-differentiation factor-15 and histamine related compounds such as histamine, amthamine, and cimetidine. Gene silencing of growth-differentiation factor-15 suppressed histamine-induced proliferation, melanin production, tyrosinase activity, and chemotactic migration of SK-MEL-2 cells. Histamine-induced expression of tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2 was also suppressed by growth-differentiation factor-15 gene silencing. On the other hand, overexpression of growth-differentiation factor-15 using a plasmid containing growth-differentiation factor-15 in SK-MEL-2 cells increased melanin production and chemotactic migration. Amthamine induced expression of growth-differentiation factor-15 in a time and concentration dependent manner. Amthamine-induced expression of growth-differentiation factor-15 was suppressed by cimetidine. Our results suggest that growth-differentiation factor-15 is a new player in histamine-induced melanogenesis, which can help researchers to extend the knowledge of the role of the transforming growth factor beta family in melanogenesis and in skin pigment disorders such as vitiligo.